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Michigan State
University

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This is to certify that the
thesis entitled
An Examination and Evaluation of Diphaloid

Potatoes of Solanum tuberosum that
Produce Mixed Modes of Zn Eggs

 

presented by

David L. Maas

has been accepted towards fulfillment
of the requirements for

 

Masters degree inPlant Breeding 8:
Genetics - Crop &
Soil Sciences

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Major professor

 

Date August 21, 1996

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

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DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU to An Affirmative AotiorVEquol Opportunity inetltwon
. mm:

 

AN EXAMINATION AND EVALUATION OF
DIHAPLOID POTATOES OP Solanum tuberosum THAT
PRODUCE MIXED MODES OF 2N EGGS

BY
David Lewis Mean

A THESIS

Submitted to
MICHIGAN STATE UNIVERSITY
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Crop and Soil Sciences
Plant Breeding and Genetics

1996

ABSTRACT

AN EXAMINATION AND EVALUATION OF
DIHAPLOID POTATOES OP Solanum tuberosum THAT
PRODUCE MIXED MODES OF 2N EGGS

BY

David Lewis Maas

Several first division restitution- (FDR) and second
division restitution- (SDR) derived tetraploid
subpopulations were constructed using two 2n egg-producing,
mixed-mode dihaploids crossed to tetraploid potatoes
(Solanum tuberosum subsp. tuberosum L.). Progeny were
separated into subpopulations using Pgm-Z segregation data.
There was no significant difference between the performance
of FDR- and SDR-derived subpopulations in multiple crosses
over multiple seasons. Inbreeding, as a consequence of the
haploidization process, may have negated any advantage of
the FDR-derived progenies over the SDR-derived progenies.
The effectiveness of the laser scanning confocal microscope
(LSCM) is demonstrated in examining megasporogenesis of
several 5. tuberosum dihaploids and diploids. Ethidium
bromide was used as a cellular stain (0.5 mg/ml for 15
minutes) to enhance the viewing of ovules. Extremely well-
defined images were obtained due to the confocal ability of
the microscope. The LSCM is powerful tool for further

observation of megasporogenesis in Solanum.

"Congress shall make no law respecting an establishment of
religion, or prohibiting the free exercise thereof; or
abridging the freedom of speech, or of the press; or the
right of the people peaceably to assemble, and to petition

the government for a redress of grievances.”
First Amendment of the Constitution

of the United States of America (1791)

iii

ACKNOWLEDGEMENTS

I would like to thank my advisor Dr. David Douches for
his patience and support during my Masters’ research. I
would also like to thank the other members of my committee,
Dr. Iezzoni and Dr. Whallon.

None of this work would have been possible without the
aid of the many spudheads who have passed through the doors
of our program. I would like to send a warm "7:00 a.m van
trip to MRF" thanks to: Andrea, Bernard, Chris, Dick Chase,
Dick Crawford, Jeff, Joe, Kate, Kaz, Kelly, Kim, Peter,
Scott and to the many undergrads that have aided me.

To my family: thanks for the support!

iv

TABLE OF CONTENTS

Page

LIST OF TABLESOOIOOOOOOOO0.0....OOOOOOOOOOOOOOOOOO0.0...

vi

LIST OF FIGURES...’I0..OOOOOOOOOOOOOOOOOOOOOO00....OOOOOViii

CHAPTER I

A STUDY TO COMPARE THE BREEDING VALUE OF FDR AND SDR 2n EGGS

IN INTERPLOID CROSSES
Introduction............................................
Literature Review.......................................
Materials and Methods...................................
Results.................................................
Discussion..............................................
Literature Cited........................................
CHAPTER II

USE OF THE LASER SCANNING CONFOCAL MICROSCOPE TO EXAMINE
MEGASPOROGENESIS IN POTATO
Introduction............................................
Literature Review.......................................
Materials and Methods...................................
Results.................................................
Discussion..............................................

Literature CitedOCOO0.00...OOOOOOOOOCCOOOOOOOOI0.0.0....

Appendix A. Environmental Data for 1991-1995
field seasons at MRF.....................

02

06

17

25

4O

49

56

58

64

67

89

94

97

CHAPTER I

Table

Table

Table

Table

Table

Table

Table

Table

Table

Table

1-1.

1-8.

1-9.

1-10.

LIST OF TABLES

Page

Representation of the number of first,
second and third order interactions for a
loci depending on allelic diversity..... 08

Genetic consequence of mode of Zn

gamete formation measured in terms of
average percent parental heterozygosity
transmitted intact to progeny............ 12

Mixed-mode dihaploids obtained from the
University of Wisconsin at Madison....... 18

Sizes of progeny populations for 2x-4x
crosses planted at MRF................... 22

Isozyme survey of dihaploids used in
2x-4x crosseSeeeeeeeeeeeeeeeeeeeeeeeeeeee 26

Tetraploid clones used in 2x-4x crosses
along with isozyme genotype.............. 27

2x-4x crosses attempted between

dihaploids heterozygous for Pym-2

or Got-2 and tetraploids homozygous

for Pgm-z or Got-2....................... 28

An examination of X2 values of the ratios
of FDR-derived progenies to the SDR-derived
progenies in 2x-4x crosses............... 30

T-test values comparing the difference
between the means for FDR- and SDR-derived
tetraploid progenies..................... 32

A summary of the total tuber yields for
2x-4x crosses in comparison to mid parent
yield values. The yields are presented

on a per plant basis..................... 33

vi

Table 1-11.

Table 1-12.

Table 1-13.

Table 1-14.

Table 1-15.

APPENDIX I

Table A-1.

Table A-2.

Table A-3.

Table A-4.

LIST OF TABLES (Continued)

A summary of average specific gravity
for three 2x-4x crosses grown at MRF
over five years in comparison to mid
parent specific gravity values...........

Skewness and kurtosis data for yield data
for 2x-4x crosses at MOOIOOOIOOOOOOOOOO

Skewness and kurtosis data for specific
gravity data for 2x-4x crosses at MRF....

Page

34

35

37

The 2n egg producing wild species available

for haplOId-SPBCIBS hYbridSe e e e e e e e e e e e e e

Percentage of FDR and SDR derived progeny
in 2x-4x crosseSOOOOOOOOOOOOOOOOOOOOOOOOO

Summary of average maximum and minimum
temperatures (in C°) during the growing
season at Montcalm Research Farm,
Montcalm, MI.............................

Summary of precipitation (in centimeters
per month) recorded during the growing
season at the Montcalm Research Farm,
Montcalm, MI.............................

Summary of soil test for the general plot
area for the growing season at Montcalm
Research Farm, Montcalm, MI..............

Summary of growing degree days (base

10 C°) during the season at Montcalm
Research Farm, Montcalm, MI..............

vii

43

45

97

98

99

100

CHAPTER 1

Figure 1-1.
Figure 1-2.
Figure 1-3.
CHAPTER 2

Figure 2-1.
Figure 2-2.
Figure 2-3.
Figure 2-4.
Figure 2-5.

LIST OF FIGURES

Page

A representation of percent parental
heterozygosity transmitted by SDR

and FDR 2n gametes in four types of
chromosomes..............................

Yield distribution of the 2x x 4x cross
4182-T x NDDZ77-2 at MRF 1991............

Specific gravity distribution of the
2x x 4x cross 4182-T x NDD277-2 at MRF

1991....0.0000IOOOOOOOOOOOOOOOOOI00......

Representation of a confocal microscope...

Ovule from 4182-T in interphase.

Nucleolus is visible and chromatin is
becoming distinct. Key: osovule,
m=megaspore mother cell

and n=nucleolus. 3000x...................

Pachynema stage of prophase shows
chromosomes more distinct and
recognizable in 84810. 9000x.............

Diplonema is characterized by the
separation of bivalents at points
along their length. 10,000x..............

During diakinesis chromosomes are

aligned in a circle on a plane with
the nucleolus of W5295.7. 8000x..........

viii

14

36

38

68

70

72

75

Figure

Figure

Figure

Figure

Figure

Figure

2-6.

2-9.

2-10.

2-11.

LIST OF FIGURES (Continued)

Page
Anaphase I, chromosomes have
begun separation to opposite poles
of W5295.7. 2000x........................ 77

Different optical section of anaphase
I shows progression of separating
chromosomes in W5295.7. 6000x............ 79

A composite was made using the numerous
optical sections provides a view of
telophase I stage in 4182-T. 6000x....... 81

A stereo reconstruction was made using
numerous optical sections provides a

view of telophase I stage in 4182-T.
ZOOOX..................................... 83

Dyad produced from 4182-T. 8000x......... 85
Functional megaspore and

three non-functional daughter cells
from 4182-T is observed. 8000x........... 87

ix

CHAPTER I

A STUDY TO COMPARE THE BREEDING
VALUE OF FDR AND SDR 2n
EGGS IN INTERPLOID CROSSES

INTRODUCTION

The cultivated potato, Solanum tuberosum subsp.
tuberosum L., is an important world food crop, ranking
fourth in total crop production (Hawkes 1990). S. tuberosum
subsp. tuberosum L. is a polysomic polyploid (2n=4x=48).
Solanum species, with ploidy levels ranging from 2x to 6x,
extend from South America to North America in a wide range
of altitudes (sea level to 4,000 meters) and climates, with
the center of diversity in the Andean region of Peru (Hawkes
1990). Wide geographical distribution has given rise to an
immense amount of diversity within native potato species.
Wild species have adaptations to environmental extremes as
well as resistances to insect pests, fungal, bacterial and
viral diseases (Hougas and Peloquin 1958; Hermundstad and
Peloquin 1985; Hawkes 1990). Other important features of
these native species are high dry matter content and low
reducing sugars. However, some may contain high levels of
toxic glycoalkaloids.

The ultimate goal in a potato breeding program is to
produce cultivars with improved horticultural
characteristics, such as increased yield, disease and insect
resistance, longer storability and desirable processing

qualities. However, there are several obstacles in breeding

2

3
potato cultivars. Most importantly, the commercial potato
is an autotetraploid (2n=4x=48), which leads to complex
inheritance patterns (Rowe 1967b). Moreover, the narrow
genetic base found in commercial cultivars is exacerbated
because many of the initial cultivars could not be used as
pollen sources due to a decline in sexual fertility
(Bradshaw and Mackay 1994, Grun et al., 1977, Mendoza and
Haynes 1974b).

Mendoza and Haynes (1974a) stated that yield in
polysomic polyploids is based mainly on intra-locus and
inter-locus interactions. The theory of maximum
heterozygosity states that hybrid vigor in cross-pollinated
polyploid species is maximized by multiple allelic
interactions at a locus (overdominance), chromosome segments
with complementary alleles (epistasis) or a combination of
both (Dunbier and Bingham 1975; Mendiburu and Peloquin
1977a; Bingham 1980; Bonierbale et al., 1993; Bingham et
al., 1994). A yield plateau was noted by Mendoza and Haynes
(1974b), where no increase in yield had occurred for the
preceding twenty years. The idea of a yield plateau was
supported by Douches et al., (1996) after comparing yield
and performance of numerous cultivars over years of
introduction. No increase in yield was observed with new
varieties (period 1970-1989) compared to varieties released
in the late 19th century.

2n gametes are an effective means of maintaining

allelic diversity and desirable epistatic interactions that

4
are needed for maximum heterozygosity and consequently
maximum expression of yield (Iwanaga 1984; Douches and
Quiros 1988a, 1988b; Concillo and Peloquin 1991). 2n
gametes can be produced through first division restitution
(FDR) or second division restitution (SDR). FDR transmits
approximately 80% of the parental heterozygosity intact,
compared to 40% by SDR (Peloquin, 1983). The use of 2n
gametes allows efficient transfer of genes from diploids to
tetraploids, as well as broadening the genetic base of the
commercial potato (Hougas and Peloquin 1958, Kotch et al.,
1992). Unilateral sexual polyploidizations utilizing 2n
gametes (4x-2x, 2x-2x, 2x—4x) resulting in tetraploid
progeny are well documented (Concillo and Peloquin 1991).
Early 4x-2x crossing studies showed a heterotic effect
associated with the use of FDR producing haploid-species
hybrids (Mok and Peloquin 1975; Mendiburu and Peloquin 1977;
Darmo and Peloquin 1990; Concillo and Peloquin 1991, Darmo
and Peloquin 1991; Ortiz et al., 1991). The heterotic
effect was also noted using dihaploids of S. tuberosum
(Werner and Peloquin 1991a, 1991c). This observed heterotic
effect is contrasted to the lower performance noted for 2x-
4x crosses which are primarily SDR. The use of separate
genotypes in the current comparison of the performance of
FDR to SDR confounds the observation. Dihaploids (haploids
derived from tetraploids, 2n=2x=24) of S. tuberosum forming
2n eggs by mechanisms genetically equivalent to FDR (delayed

meiotic division) and SDR (delayed meiotic division,

5

omission of second division) have been identified (Werner
and Peloquin 1991c). The use of these mixed mode producing
dihaploids allows for an unbiased comparison of FDR and SDR.

The objective of this research is to conduct field
studies to compare the performance of FDR- and SDR-derived
tetraploid progeny populations. With the ability to
separate the progeny into SDR- and FDR-derived
subpopulations we are able to make unbiased comparisons of
the value of differing modes of Zn gamete formation. These
evaluations may also give us insights into the role of
maximum heterozygosity and the expression of heterosis in

autotetraploid potato.

LITERATURE REVIE'

MAXIMUM HETEROZYGOSITY THEORY

Numerous studies have been conducted to study and
understand the genetic basis for yield in potato and other
polyploid crops. Allopolyploids or disomic polyploids can
maintain heterozygosity in two or more divergent genomes.
Thus, allopolyploids do not suffer inbreeding depression due
to the internal hybridity among their loci. In contrast,
autopolyploids or polysomic polyploids must rely upon cross-
pollination to ensure intra-locus heterozygosity (Bingham
1980).

Rowe (1967a) examined the performance of diploid potato
hybrids (S. tuberosum dihaploid x S. phureja) and
vegetatively doubled diploids. Tuber production of
tetraploids was inferior to that of the diploids. Rowe
postulated that if heterozygosity influences plant yield and
doubling of the diploids produces genotypes with a
restricted number of heterozygous loci, inbreeding may
result. In later research, Rowe (1967b) compared the
performance of 11 diploid and autotetraploid families. The
hybrids originated from crosses between diploid potato
hybrid (S. tuberosum dihaploid x S. phureja) and

7
vegetatively doubled diploids. Diploid performance was less
than their parental clones, in contrast, the tetraploids
exceeded the midparent value as well as the higher parent
(Rowe 1967b). Rowe concluded that for productivity in
diploid and tetraploid potatoes, gene action and
heterozygosity may be more important than ploidy level. De
Jong and Rowe (1971), in additional crossing studies using
diploid species S. phureja, S. stenotomum and dihaploids of
S. tuberosum, found that 8,, S2 and S, progenies exhibited a
linear relationship between the decrease in tuber production
and the expected coefficient of inbreeding in successive
generations of selfing.

Mendoza and Haynes (1974a) explained heterosis for
yield by a model of overdominant gene action instead of
dominant gene action based on Rowe’s (1967a,b) data. With
overdominance (intra-locus interactions), autotetraploid
genotypes with high frequencies of tetra- or triallelic loci
(more first-order interactions) are superior to those
genotypes with higher percentage of di- or monoallelic loci
(fewer or no first-order interactions) (Bingham 1980,
Bonierbale et al., 1993) (Table 1-1).

To further explore the importance of multiple allelic
interactions, researchers in alfalfa (Megicago sativa L.
sensu lato) generated haploid-derived autotetraploids (HD4x)
(Dunbier and Bingham 1975). Use of HD4x provides a model
system where populations are of a defined genotypic

structure. Single crosses (SC) between unrelated HD4x were

Table 1-1. The number of first, second and third
order interactions for a locus depending on allelic
diversity.

 

Number of interactions

 

 

 

Locus First Second Third
Tetra-allelic

(AIM) 6 4 1
Tri-allelic

(AIAIAzAa) 3 1
Di-allelic

(AIAIAIAQ) / (A1A1A2A’Z) 1

Mono-allelic

(AIAIAIAI) 0

 

9
made, subsequent double crosses (DC) were performed using a
random selection of SC plants. Resulting forage yields,
seed weight and fertility data show that the DC was
consistently greater than the SC. This result supports the
importance of multiple allelic interactions and the
hypothesis that maximizing heterozygosity maximizes yield in
alfalfa.

Alfalfa is a cross-pollinated autotetraploid species,
although self-pollination is possible. In alfalfa, vigor
and fertility declined more quickly than the theoretical
rate of reduction of heterozygosity during selfing (Busbice
and Wilsie 1966; Busbice 1968). Studies with inbreeding
have shown a decrease of seed yield of half when the
inbreeding coefficient was 0.06 and 5/6 when the coefficient
was 0.30 (Busbice 1968). Busbice explained these
observations by epistatic interactions of recessive lethals
masking the effects of fertility genes.

Bingham (1980) proposed the theory that maximum
heterozygosity in autopolyploids is due to the presence of
multiple alleles at a locus in combination with chromosomes
containing linkage blocks (epistasis). Epistasis (inter-
locus interactions) occurs when dominant alleles at
different heterozygous loci complement each other by masking
recessive deleterious alleles. Bonierbale et al. (1993)
developed several papulations to test the theory. One
population consisted of a cross between two adapted S.

tuberosum cultivars (TT). The second was produced by the

10

intermating of a Neotuberosum-Tuberosum hybrid by S.
tuberosum spp. andigena (NT). A cross between a S.
tuberosum advanced selection and a S. tuberosum x (S.
phureja-haploid S. tuberosum hybrid) provided the third
population (PT). Restriction fragment length polymorphism
data showed that the TT population had the highest level of
homozygosity (29% vs 24% for NT and 22% for PT). Relative
heterozygosity index showed PT to be higher than either NT
or TT (50% vs 48% and 47%). Performance trials showed that
in adapted x adapted crosses, homozygosity was negatively
correlated with tuber yield and maximum heterozygosity was
positively correlated to yield for tubers over 6.4 cm.
However, crosses between adapted and unadapted parents
showed no relationship between yield and heterozygosity.
These results are explained by the idea that maximum
heterozygosity only influences tuber yield in a favorable
direction when adapted parents are combined. An alternate
hypothesis could be the effect of negative epistatic
interactions. Genes for lack of adaptation could be masking
alleles in adapted material producing no increase in mean
performance. It has been noted in group Phureja, yield
increased over generations as populations became long-day
adapted (Bradshaw and Mackay 1994).

Another method to examine maximum heterozygosity is
through the use of Zn gametes. 2n gametes can be produced
by different mechanisms each with different genetic

consequences: 1) premeiotic chromosome doubling; 2) first

11
division restitution (FDR)(parallel spindles, synaptic
variant, delayed meiotic division); 3) second division
restitution (SDR)(delayed meiotic variation, omission of
second division, irregular anaphase II, no second
cytokinesis); 4) post meiotic chromosome doubling; 5)
apospory (development of the gametophyte from a somatic
cell) (Rhodes and Dempsey 1966; Mendiburu and Peloquin 1975;
Hermsen 1984; Veilleux 1985; Ramanna 1979; Werner and
Peloquin 1991b). Each mode of Zn gamete formation transmits
a different amount of parental heterozygosity intact to the
progeny (Table 1-2)(Werner 1989). Several meiotic mutants
leading to 2n pollen and egg production have been identified
in potato (Mok and Peloquin 1975b; Iwanaga and Peloquin
1979; Iwanaga 1984; Stelly and Peloquin 1986; Werner and
Peloquin 1987; Douches and Quiros 1988b; Ramanna 1979).
Dihaploids of S. tuberosum forming 2n eggs by mechanisms
genetically equivalent to either FDR (synaptic variant,
delayed meiotic division) or SDR (omission of second
division, irregular anaphase II, no second cytokinesis,
delayed meiotic division) have been identified (Werner and
Peloquin 1991c). Use of both forms of Zn gametes in
interploidy crosses (2x-4x, 4x-2x and 2x-2x) is well
established (Mok and Peloquin 1975a, 1975b; Mendiburu and
Peloquin 1977a, 1977b; Peloquin et al., 1989; Darmo and
Peloquin 1990; Concillo and Peloquin 1991; Darmo and
Peloquin 1991; Ortiz et al., 1991; Ortiz and Peloquin 1991).

Studies performed by Peloquin and his co-workers have led to

12

Table 1-2. Genetic consequence of mode of 2n
gamete formation measured in terms of average
percent parental heterozygosity transmitted
intact to progeny.

 

 

Mode of Zn gamete formation Heterozygosity
transmitted
Post meiotic chromosome doubling 0%
Second division restitution 40%
Premeiotic chromosome doubling 62%
First division restitution 80%

Apospory 100%

 

13
the hypothesis that FDR-derived 2n gametes are superior to
SDR-derived 2n gametes with respect to the performance of
resulting tetraploid progeny (Mendiburu and Peloquin 1977b;
Ortiz et al., 1991; Werner 1989; Werner and Peloquin 1991c).
Higher tuber yields have been found in FDR-derived
tetraploids than in SDR-derived tetraploids from 4x-2x
matings using haploids and haploid-species hybrids
(Mendiburu and Peloquin 1977a, 1977b; Chujoy and Peloquin
1986; Darmo and Peloquin 1990; Concillo and Peloquin 1991;
Darmo and Peloquin 1991; Ortiz and Peloquin 1991; Werner and
Peloquin 1991a, 1991c). Heterotic responses from 4x-2x
(FDR) matings with these clones showed progeny yields that
surpass midparent values and tetraploid controls in yield
trials. Increased yield production of the 4x-2x populations
was attributed to the high level of heterozygosity
transmission through FDR pollen. FDR 2n gametes transmit
approximately 80% of their parental heterozygosity intact to
the progeny compared to 40% by SDR 2n gametes (Figure 1-1)
(Werner and Peloquin 1987; Peloquin et al., 1989; Concillo
and Peloquin 1991; Darmo and Peloquin 1991). In contrast,
Mendiburu and Peloquin (1977a), attributed low yield of
progeny derived from 2x-4x crosses to the type of 2n egg
formation. The primary mechanism of Zn egg production in
potato is SDR. This furthered the belief that the breeding
value of FDR-derived 2n gametes are indeed superior to SDR-
derived 2n gametes.

Bingham et al. (1994) reinterpreted the research of

14

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15
Dunbier and Bingham (1975), attributing apparent multiple
allelic interactions in previous studies to linkage
disequilibrium. The reexamination of Dunbier and Bingham
(1975) and other studies concluded that progressive
heterosis in autotetraploids is due to a increase in
complementary gene action (Bingham et al. 1994).
Populations involving two alleles per locus (chromosome
doubled diploids) supported that the accumulated action of
favorable alleles not intra-allelic interactions improved

performance in alfalfa (Bingham et al. 1994).

HALF TETRAD ANALYSIS

A unique advantage of 2n gametes is the opportunity
they offer for half-tetrad analysis (HTA)(Mendiburu and
Peloquin 1979; Douches and Quiros 1987; Douches and Quiros
1988a; Werner et al., 1991). HTA is based on the fact that
two strands of a bivalent from the diploid parent are
recovered in the tetraploid progeny. HTA is feasible in a
2x-4x cross in which a diploid, heterozygous for the
codominant marker proximal to the centromere, is crossed to
a monoallelic (nulliplex) tetraploid. For FDR-derived
progeny the markers will all be simplex (Aaaa), if the
progeny are SDR-derived one half of the markers will score
as nulliplex (aaaa) with the balance duplex (AAaa). HTA is
useful in the determination of the ratio of FDR and SDR
gamete production in mixed-mode progeny if a marker locus is

tightly linked to the centromere. Dihaploids of S.

16
tuberosum forming 2n eggs by mechanisms genetically
equivalent to FDR (delayed meiotic division) and SDR
(delayed meiotic division, omission of second division) have
been identified (Werner and Peloquin 1991c). The use of
separate genotypes in previous comparisons of the
performance of FDR to SDR confounds earlier observations
(Mendiburu and Peloquin 1977a, 1977b; Chujoy and Peloquin
1986; Darmo and Peloquin 1990; Concillo and Peloquin 1991;
Darmo and Peloquin 1991; Ortiz and Peloquin 1991; Werner and
Peloquin 1991a, 1991c). To avoid confounding, the use of
these mixed-mode producing dihaploids allows for an unbiased

comparison of FDR and SDR.

17

MATERIALS AND METHODS

1. Plant Material

Dihaploids (2n=2x=24) previously identified as mixed-
mode 2n egg producing, derived from tetraploid S. tuberosum
(Werner, 1989), were kindly provided by Drs. Werner and
Peloquin (University of Wisconsin, Madison)(Table 1-3).
Tetraploid clones of potato cultivars (Lemhi Russet, Ranger
Russet, Frontier Russet, Ontario, Norgold Russet, Yukon
Gold, Chippewa and Desiree) and breeding lines (NDD277-2,
ND860-2, and LA12-59) were obtained from the Michigan State

University potato breeding program.

2. Isozyme analysis

Crude protein extracts were obtained by crushing 150 mg
of fresh juvenile potato leaf tissue from the greenhouse.
Before crushing, 150 pl of 0.1 M tris-HCL buffer, pH 7.8,
with 2% glutathione was added to the sample to reduce
oxidation. Each sample was absorbed into 2 x 10 mm wicks of
3 MM Gel Blot Paper (Schleicher and Schuell, Keene, NH.),
placed into plastic racks, then wrapped in Saran Wrap and
stored at -20°C overnight.

Electrophoretic assays were performed using a

18

Table 1-3. Mixed-mode dihaploids
obtained from the University of
Wisconsin, at Madison.

 

 

Dihaploid Maternal Source
4182-T W231

H175 Redsen

H57 W231

H69 Merrimack

H76 Merrimack

H109 Merrimack

H115 Merrimack

H161 W760

 

19
tris-citrate, pH 8.3 gel buffer system (Vallejos, 1983).
The gel matrix consisted of 10 % hydrolysed potato starch
(STARCHart, Corp., Smithville, TX). Starch gels were left
at room temperature (23°C) overnight and loaded the
following morning. Wicks were loaded into refrigerated gels
(4°C) along with a marker lane (bromophenol blue) and
electrophoresed at 50 mA until the marker had run within 2
cm of the anodal end of gel. Gels were allowed to cool for
10 minutes and sliced. Slices were stained for either
phosphoglucomutase (PGM) or glutamate oxaloacetate

transaminase (GOT) according to Vallejos (1983).

3. Parental classification

Two segregating loci were used for HTA: Pym-2 and th-
2 which are each 2.0 cM from the centromere, located on
chromosome 4 and 7, respectively (Douches and Quiros 1987).
Dihaploids were surveyed to determine their isozyme genotype
for these loci. Only dihaploids heterozygous for Pgm-Z or
Got-2 were selected for use in 2x-4x crosses. Tetraploid
cultivars that are nulliplex at least at one of two loci

(Douches and Ludlam 1991) were selected for 2x-4x crossing.

4. 23-4: crosses

Sprouting tubers of S. tuberosum dihaploids and
tetraploids were planted in the greenhouse for crossing. To
promote flowering, petri dishes were put in twelve-liter

plastic pots three-fourths full with Baccto mix in the

20

greenhouse in January of each year. Tubers were covered
with Baccto mix until the roots had grown over the petri
dish into the lower portion of the pot. The Baccto mix was
then washed away from the tubers to expose stolons.
Placement of the tubers on the petri dishes allowed for the
easy removal of stolons and tubers. This practice promoted
continuous flowering. Shoots originating from the tubers
were thinned to 1-2 stems, trimmed weekly, and allowed to
flower. Pollen from the tetraploid parents was collected in
gelatin capsules and stored at 4°C for up to one month until
use. From 1990 to 1993, flowers on the dihaploids were
emasculated and pollinated with tetraploid pollen from March
through May. High pressure sodium lights were used as
supplemental lighting (16 hr daylength). The greenhouse
nighttime temperature was maintained at 25%L

Seeds were extracted from mature fruits and dried.
Prior to planting seeds were treated with 1500 ppm GA3 for
24 hours. Treated seeds were placed in 15 cm pots filled
with Baccto potting mix and covered lightly. Once
germinated seedlings had produced true leaves, they were
transplanted into cell packs. Seedlings were assayed for
Pym-2 and Got-2 at the four to five leaf stage to classify
individuals as SDR- or FDR-derived progeny. The most
juvenile leaf was taken from each seedling for crude protein
extraction. Seedlings in cell packs were allowed to mature
and tubers from each seedling were harvested then held at

4°C until the planting season.

21

5. Progeny classification

' Tetraploid progenies were separated into FDR- and SDR-
derived populations based on the isozyme genotype of the
Pgm-z and cot—2 loci, (Douches and Quiros 1988a,1988b;
Werner et al., 1991). The expectations of the 2x-4x
segregation is as follows: the FDR-derived progeny are
simplex (Aaaa), while the SDR-derived progeny are either

duplex (AAaa) or nulliplex (aaaa).

6. Field studies

Field experiments were conducted from 1991 to 1995 at
the Montcalm Research Farm (MRF), Montcalm, MI. Plots were
planted with 30 cm spacing between hills with 102 cm between
rows. Progeny and parents from the three crosses (Table 1-
4) were planted in a randomized complete block design
(RCBD). Individuals with sufficient micro-tubers from the
progeny classes and parents were planted as four-hill plots
‘with each progeny individual replicated three times and each
parent replicated 10 times. Harvested tubers from the

current year were used as seed within each papulation the

following year.

7 . Cultural

The soil in the experimental plots at MRF is a McBride
Sandy Loam with a 2% slope. Initial soil tests showed a
‘very good base nutrition value for the research plots at MSU

(Table A-1). Additional fertilizer was applied as needed

22

 

 

 

 

omna ntoa amim mm nv oaou coca; on mean mmma
cmna mica amim an am Nloowoz x Blmwaw mmma
mama naim warm an am «loomoz x Bimmae cmma
vwma eatm «aim ooa mm Nthbmoaz x Bimmaw nmma
mmaa amim main oaa mm Nlhhuooz x Bimmaw Nmma
coma naim calm oma am Nthhmooz x Bimmav amma
56:3 3.3 3.3 com mom 39.8 new»

somcmm undersea Osauccam

Guam
>cmmoum

.mmaau

oa mundane may use mesa» conga coucoaamcu aosca>aGCa mammoua some
spas uuoam aaanincou me vmuccam one: mucmuom use mmmmoao anemone
on» scum macsma>accH .omom c :a mm: as couscam cums mcoaucasmoa
aa¢ .uemnouo xvixm you mcoaucacmom anemoun no nouam .via canes

23
during the growing season. Approximately 327 kg/Ha 0-0-
60(N:P:K) was applied at plowdown, 218 kg/Ha 20-10-20 was
applied during planting, 218 kg/Ha 34-0-0 broadcast at
emergence, 109 kg/Ha 46-0-0 sidedress at hilling and 130
kg/Ha 28-0-0 through irrigation. Herbicide treatments
(metalochlor 2.2 kg/Ha with metrabuzin 0.6 kg/Ha) were
applied preemergence. Hilling was done when plants were 25-
30 cm in height. Irrigation was applied according to best
management practices (Table A-2). Overall total for natural
precipitation varied considerably over four years (Table A-

3).

8. Harvest

A single row chain harvester was used to lay the tubers
on the soil. Tubers were collected and bagged by hand on a
per plot basis. For all field plots a red-skinned cultivar
(Red Norland or Red Pontiac) was placed between plots to aid
in separation at harvest. Vines were killed with Diquat

(one pint/acre) two weeks prior to harvest.

9. Yield and Specific Gravity Evaluation

Total tuber yield was measured from tubers of each plot
and was weighed using digital scales accurate 1 1 gram
(Toledo Scale, Worthington, OH). Stand counts were taken
for the four-hill plots one month after emergence. Yield
data was adjusted with the stand count data to present

yields on a per plant basis. A 0.5 to 4.0 kg sample from

24
each plot was taken for specific gravity measurement.
Specific gravity was calculated as the dry weight of the
sample divided by the difference of tuber weight in air

minus weight in water.

10. Statistical Analysis

Mean separations (least significant difference) and t-
tests of population means and variances for the progeny
classes were performed using MSTAT-C (Michigan State
University). Skewness and kurtosis values were examined for
both yield and specific gravity to characterize the

distribution of progeny classes.

25

RESULTS

1. Isozyme results of dihaploids and tetraploids and 22-4:
progeny

Five of eight dihaploids were heterozygous for Pym-2
and 6 of eight were heterozygous for Got-2 (Table 1-5).
Twelve cultivated tetraploid potatoes and breeding lines
were homozygous for Pgm-z. Four of the twelve were
homozygous for Got-2 locus (Table 1-6).

Crosses were attempted using combinations of a subset
of all available dihaploids and tetraploids (Table 1-7).
Only seven crosses out of a possible 66 combinations
provided viable seed. Progeny from all successful 2x-4x
crosses were genotyped for Pgm-z and Got-2. For Pym-2, all
progeny from the dihaploid 4182-T fit a 2:1 ratio of SDR:FDR
whereas those from dihaploid H175 had ratios that fit a 1:1
(Table 1-8). For Got-2, progeny from H175 x Yukon Gold did
not show ratios that supported the Pym-2 isozyme data and
therefore Got-2 was not used to classify progeny. Crosses
providing sufficient progeny numbers were used in field

experiments.

26

Table 1-5. Isozyme survey
of dihaploids used in 2x-4x
crosses.

 

 

 

Locus
Dihaploid Pgm-z Got-2
H175 23' 35b
H115 23 35
H69 23 35
H76 22 35
H57 22 35
H161 23 35
4182-T 23 33
H109 23 35

 

‘ 23 denotes Pgm 222’
' 35 denotes Got 2’25

27

Table 1-6. Tetraploid clones
used in 2x-4x crosses along
with isozyme genotype.

 

 

 

Locus
Tetraploid Pym-2 Got-2
NDD277-2 2222' 3355'
ND860-2 2222 3355
LA12-59 2222 3355
Lemhi R. 2222 3555
Norchip 2222 3335
Ranger R. 2222 3555
Frontier R. 2222 3555
Ontario 2222 3355
Norgold R. 2222 5555
Yukon Gold 2222 5555
Chippewa 2222 5555
Desiree 2222 5555

 

' 2222 denotes Pgm-Z 22222222
" 3355 denotes Got-2 23232525

28

Table 1-7. 2x-4x crosses attempted between
dihaploids heterozygous for Pgm-Z or Got-2
and tetraploids homozygous for Pgm-Z or Got-2

 

Dihaploid (2x) Tetraploid (4x)

 

4182-T X NDD277-2
4182-T x N0860-2

4182-T x LA12-59

4182-T x Lemhi Russet
4182-T x Norchip

4182-T x Ranger Russet
4182-T x Frontier Russet
4182-T x Ontario

4182-T x Norgold Russet
4182-T x Yukon Gold
4182-T x Desiree

H175 X NDD277-2

H175 x ND860-2

H175 x LA12-59

H175 x Lemhi Russet
H175 x Norchip

H175 x Ranger Russet
H175 x Frontier Russet
H175 x Ontario

H175 x Norgold Russet
H175 x Yukon Gold
H175 x Desiree

H115 x NDDZ77-2

H115 x ND860-2

H115 x LA12-59

H115 x Lemhi Russet
H115 x Norchip

H115 x Ranger Russet
H115 x Frontier Russet
H115 x Ontario

H115 x Norgold Russet
H115 x Yukon Gold
H115 x Desiree

Table 1-7.

(Continued)

29

 

Dihaploid (2x)

Tetraploid (4x)

 

H161
H161
H161
H161
H161
H161
H161
H161
H161
H161
H161
H109
H109
H109
H109
H109
H109
H109
H109
H109
H109
H109
H69
H69
H69
H69
H69
H69
H69
H69
H69
H69
H69

KKKKNXKXXXXKKKKXXKKKKXKXXXXKKKXKK

NDDZ77-2
ND860-2
LA12-59

Lemhi Russet
Norchip

Ranger Russet
Frontier Russet
Ontario
Norgold Russet
Yukon Gold
Desiree
N00277-2
ND860-2
LA12-59

Lemhi Russet
Norchip

Ranger Russet
Frontier Russet
Ontario
Norgold Russet
Yukon Gold
Desiree
NDD277-2
ND860-2
LA12-59

Lemhi Russet
Norchip

Ranger Russet
Frontier Russet
Ontario
Norgold Russet
Yukon Gold
Desiree

30

.mOaucu oouocfloua :0 ocean naosoa>acca no nonfisz .
.oc>aumvimom we coauauuoao maosoa>acca no Hmnasz .
.vc>aumcimom me omauanuoau uaocoa>aUCa uo Monasz .

 

 

 

 

 

 

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ao.o me.o -.e an.o ee.m nn.ma m.ea m.ea ea ma «unemaoz mean
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e.o a.o ea.a ~m.a nn.ea no.4n em on an an «noemoz eamwae
ms.o moo.o o~.o m~.m so.o~ nn.ae an an ma me .m ummcmm summae
m.o aoo.o ao.o n~.ma nn.oo so.o~a m.om m.om am oma albumen: summav
m.e mo.o ao.o sm.e nn.~a he.e~ m.wa m.ma ~a mm .a aasmq simmae
a u u a u a a a u a a a .mou .moe Ros Roe .mom .soe caoamauuoe 303nm
noun» space a»« aua menace essence
a as manage museums» onemmemo

 

.mmmmono Reina ea mmaccooum oe>aumolmom

on» on moan—000.3 om>aumoimom mo moans.» on» no mosacer «x no coauccaficxc 5a .mia canoe

31

2. Field experiments

For three years (1991, 1992, 1993) no difference was
seen for total tuber yield or specific gravity of FDR- and
SDR-derived tetraploid progeny from the cross 4182-T x
NDD277-2 (Table 1-9). No difference in yield or specific
gravity between the FDR- and SDR-derived populations was
observed in the cross 4182-T x ND860-2 in 1994 and 1995.
Similar results were found in the H175 x Yukon Gold in 1995.
The average yields for the three FDR- and SDR-derived
populations approached the mid-parent values during most of
the years (Table 1-10). Specific gravity values did not
surpass mid-parent values most of the years (Table 1-11).

The distributions of FDR- and SDR-derived populations

are demonstrated by the kurtosis values (Tables 1-12, 1-13).
A positive value denotes a peaked curve whereas a negative
value represents a flatter curve. Kurtosis values for yield
(Table 1-12) did not follow any trends. This is typified by
the cross 4182-T x NDD277-2 for 1991, 1992 and 1993. In
1991, the SDR-derived progeny had a tighter distribution
around the mean. During 1992 there was no difference
between the distributions, whereas 1993 showed the FDR-
derived progeny with a significantly tighter distribution
around the mean. A sample yield distribution is shown for
4182-T x NDDZ77-2 during 1991 (Figure 1-2). Data for the
specific gravity distribution was similar to that of the
yield values and did not follow trends (Table 1-13). Figure

1-3 shows the specific gravity distribution for 4182-T x

32

Table 1-9. T-test values comparing the

difference between the means for FDR-
and SDR-derived tetraploid progenies.

 

 

Specific
Year Cross Yield‘ Gravity‘
1991 4182-T x ND0277-2 0.9210 ns 0.7145 ns
1992 4182-T x NDD277-2 0.1984 ns 0.4796 ns
1993 4182-T x NDD277-2 0.8965 n8 0.3707 n8
1994 4182-T x ND860-2 0.8147 n8 0.0642 n8
1995 4182-T X ND860-2 0.4390 n8 0.4998 ns
1995 H175 x Y. Gold 0.8042 ns 0.6648 n8

 

‘ Populations within a year are not

significantly different from each other

at P s 0.05.

33

Table 1-10. A summary of average of tuber
yields (gms/plant) for three 2x-4x crosses
grown at MRF over five years in comparison
to mid parent yield values.

 

 

Year Cross Group Yield
1991 4182-T x NDD277-2 FDR 1371
SDR 1527
Mid Parent 1360
1992 4182-T x NDD277-2 FDR 482
- SDR 542
Mid Parent 639
1993 4182-T x NDD277-2 FDR 551
SDR 554
Mid Parent 414
1994 4182-T x ND860-2 FDR 1085
SDR 1062
Mid Parent 1224
1995 4182-T x N0860-2 FDR 1282
SDR 1179
Mid Parent 1182
1995 H175 x Y. Gold FDR 1086
SDR 1129

Mid Parent 931

 

34

Table 1-11. A summary of average specific
gravity for three 2x-4x crosses grown at
MRF over five years in comparison to mid
parent specific gravity values.

 

 

Specific

Year Cross Group Gravity
1991 4182-T x NDD277-2 FDR 1.074
SDR 1.074
Mid Parent 1.078
1992 4182-T x NDD277-2 FDR 1.076
SDR 1.075
Mid Parent 1.082
1993 4182-T x NDD277-2 FDR 1.055
SDR 1.054
Mid Parent 1.058
1994 4182-T x NDB60-2 FDR 1.065
SDR 1.062
Mid Parent 1.076
1995 4182-T x N0860-2 FDR 1.065
SDR 1.064
Mid Parent 1.064
1995 H175 x Y. Gold FDR 1.069
SDR 1.068

Mid Parent 1.071

 

35

aaoe.e an unnoauaemam mauoemc «a;
uac.o us unscauacoau ucuocmo as
ame.e an nemoauaemau mouoemc «

assuauacmamcoc ucuoccc us.

 

 

 

 

menu mmu Immhé aammmé mom oaoo .» x mmaz
aamN awn . 235.0 .mmcm.o mom Uaoo .u on mean mmma
mama new Immm.o .tonma.a mom Nlomwoz x BINmae
mama con 233.0! Iombm.o mam Ntowwoz x Btmmav mmma
omnu omn iaomé :hmova mam mlcwmoz x ulnmav
oenn con Imvo.al aonvn.o MD."— Nloomoz x Btmmav cmma
omom one ImNB.OI Ioovn.o mom Nathan—z x Elmmae
0:.N onN .8wmm.a :.oah~.a mom Nthhmoaz x filmmaw nmma
omnn nos lmmo.o :Nwmhév mow Nibbmnoz x Blmwae
NmNN mnn Iman.o :woam.o mom Nthhmnoz x Blmmav Nmma
mnmn can. .:eee.e .:e~em.a mom cinemas: x sameae
mNon who Imnm.ot amanm.a mom airman—OZ x Blmmav amma
xcz Ca: .uauouusx .mmcgcxm coo: macho no";
mmccm
.mm: as someone Xeixm
you even mama» How mesao> manouusx use mumcsoxm .Nala manna

36

 

 

to“. B

mom I

 
 

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z, z .
2
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or

'I'DPIMDW
1° M

9

37

aaoo.o us unscauacmau neuocec as;

wao.o us unscauacmau umuocec
«mo.o us unscauacmau neuocmc

i!
t

acooauacmamcoc monococ mm.

d

 

omo.a amo.a lows-.0 amhmvéu mom UaOU .w x wham

mmo.a omo.a amem.o avmao.o... mam UaOO .> x mbam mmma

wmo.a amo.a INnm.o ..nm~..o.a mom Nlowwaz x Btnmav

omo.a mno.a 8N¢O.O aooewéu Mam Nlommn—z X Btflmaw mmma

th.a omo.a Inwa.ot lmamo.o mom Nlowmoz x Blmmav

cmo.a amo.a Ibww.o annmm.o mom Ntoomn—z X Eleav Mvmma

nmo.a anota .SMNO.N ...hmmm.o Mam Nlthn—Oz x BtNmav

omo.a anota INVOA. awov¢.o mom NithDn—z X Bimwav nmma

wmo.a weo.a ammo.o ahmma.0l mam Nibbflaaz x filmmaw

ooa.a Neota :hnm.a :.vh¢o.a mam mlthn—Dz x Binmaw mmma

moa.a omo.a Imhh.o amona.o Mom Nithn—Dz x Blflmav

mmo.a mwo.a .8mha.h .:o~om.al mom «lbs-N092 x Bimmav amma
an“: Had” an .m mOanaan emwmgmxm mg: MMOHU HMO N

 

omccm

 

>Ha>eum cauaoemu you mesao> mauouusx use uncssexm

.mmz us uemmouo vaXN you sumo
. nala Danna.

4182-T x NDDZTI-Z

38

 

I SDR

fl FDR

 

 

 

 

 

 

 

 

 

z/y

 

 

 

 

 

 

1 .M-1 .oss

 

1 .001 .0“

 

1.11011”

\\ :x\ \
moo-1.009

1.0 70-1 .070

1 .6-1 .00

1 mm .030

Speclflcgrevly

Fiat-e 1-3. Speclflcgevlyddebrm4182-TXNDD277-2MMRF1991.8pedflcgreviydubstwnon
epsrpiotheels.

39
NDD277-2 during 1991.
Several crosses (4182-T x Lemhi Russet, 4182-T x Ranger
Russet, 4182-T x Yukon Gold and H175 x NDD277-2) did not
survive the initial field season in large enough numbers to

establish a replicated study, therefore were not used

further.

40

DISCUSSION

The lack of superiority of FDR-2n eggs evident in this
study contradicts previously published results. Studies
examining the performance of FDR-derived 2n eggs in
comparison to SDR-derived 2n eggs found that the FDR-derived
progeny were superior in 4x-2x crosses utilizing both
dihaploids and dihaploid-species hybrids (Mendiburu et al.,
1974; Mendiburu and Peloquin 1977b; Chujoy and Peloquin
1986; Tai 1987; Werner 1989; Ortiz et al., 1991; Werner and
Peloquin 1991c). In all previous cases the FDR-derived
tetraploid progeny came from different crosses than the SDR-
derived tetraploid progeny. Thus, the 2x genotype and the
mode of Zn egg formation was confounded, producing a bias.
If a well adapted FDR pollen producing clone is used in
comparison to a less-adapted SDR egg producing clone there
may be yield differences masking the superiority of the mode
of Zn gamete formation. Through the use of mixed-mode
dihaploids we were able to produce both FDR and SDR 2n
gametes from the same genotype, allowing us to eliminate
confounding by the parental genotype with the mode of 2n
gamete formation.

A possible contributing factor for the lack of

difference between the FDR- and SDR-derived tetraploids may

41
be the level of heterozygosity in the dihaploid parent used.
Dihaploids produced from S. tuberosum tetraploids by
haploidization, have a measure of inbreeding. It has been
estimated that the haploidization process is equivalent to
three generations of inbreeding (Yeh et al., 1964; Howard
1970; thewaal 1987). This inbreeding reduces the level of
heterozygosity in the dihaploid and leads to the loss of
intra- and inter-locus interactions. As a consequence,
drastic yield declines, loss of vigor and fertility can
occur (Busbice and Wilsie 1966; Dewey 1966; Levings et al.,
1967; Busbice 1968; Dessureaux and Gallais 1969; Rowe 1967a;
Mendiburu et. a1 1974; Mendoza and Haynes 1974a; Rice and
Dudley 1974; Bingham 1980). In all dihaploids, a lack of
vigor, low yields and infertility were noted in comparison
to tetraploids (data not shown). To determine whether the
level of inbreeding in the dihaploid was the factor in not
providing the previously observed superiority of haploid and
haploid-species hybrid FDR-derived progenies, the allelic
diversity of the mixed-mode parents used in 2x-4x crosses
should be increased. Crossing the dihaploids to Solanum
species would increase the allelic diversity of the
resulting dihaploid-species hybrids (D-SH) (Hermundstad and
Peloquin 1985; Darmo and Peloquin 1990; Hawkes 1990;
Concillo and Peloquin 1991; Darmo and Peloquin 1991). These
D-SH could be used to construct genetically diverse 2x-4x
populations. A series of mixed-mode producing Solanum

species previously identified by Werner (1989) would be

42
suitable for this purpose (Table 1-14). An alternative to
constructing D-SH is to increase the allelic diversity of
the tetraploid clones prior to producing dihaploids.
Molecular evidence has provided information on levels of
allelic diversity in potato. A survey of 10 isozyme loci
from 40 Solanum species showed wide diversity between
species (5.7 alleles/locus)(Douches et al., 1989). In
contrast, isozyme analysis of North American potato
cultivars for the same loci show that allelic variation is
2.15 alleles/locus and that the majority of loci in the
cultivars are diallelic (Douches and Ludlam 1991). The
possibility of low levels of allelic variation in polyploids
is supported by research in blueberries (Hokanson 1996).
Potato restriction fragment length polymorphism (RFLP)
analysis showed that S. tuberosum was the least heterozygous
when compared to S. tuberosum spp. andigena and S. phureja
(Bonierbale et al., 1993). The fact that few tri- or tetra-
allelic loci have been observed in cultivated potato, yet
heterosis is seen regularly, may suggest epistatic gene
interaction might be contributing to performance. If we
assume that tetraploid potato is diallelic at most loci,
this leaves only one first order interaction between
alleles. In this light, heterosis would more likely come
from the effect of complementary gene action, supporting
Bingham's reevaluation (1994).

Pgm-Z, a proximal locus located 2.0 cM from the

centromere, was used to separate the progeny classes. A

43

Table 1-14. The 2n egg producing wild species
available for haploid-species hybrids.

 

 

Solanum Accession Modes
Species Number‘

S. brevicaule 233961 FDR/SDR
S. sparsipilum 230502 FDR/SDR
S. sparsipilum 310933 FDR/SDR
S. tarijense 414148 FDR/SDR

 

' P.I. Numbers from Potato Introduction Station,
Sturgeon Bay, Wisconsin.

44
certain degree of misclassification is expected due to
recombination. With a population size of 181 (4182-T x
NDD277-2) assuming a 2% misclassification rate, 1 FDR-
derived and 2 SDR-derived progenies would be misclassified
(Werner et al., 1991). If a second proximal locus is
available for HTA the amount of misclassification can be
reduced. Dihaploid parents 4182-T and H175 are heterozygous
for Pym-2 with H175 also heterozygous for the Got-2 locus.
Tetraploid progeny from the cross H175 x Yukon Gold were
assayed for both Pgm-Z and Got-2. Got-2 results indicate a
more distal gene-centromere estimate then with the previous
gene-centromere mapping estimate (Douches and Quiros 1988).
The previous gene-centromere map distance for Got-2 was
determined from a haploid-species hybrid. Gene-centromere
distances may have been underestimated due to reduced
recombination in the hybrid (Douches and Quiros 1988).
Therefore, Got-2 segregation data was not used to classify
the 2x-4x progeny in this study.

The ratio of FDR-derived progenies to the SDR-derived
progenies observed for the 2x-4x crosses in the experiment
was dependent on the dihaploid parent used (Table 1-7).
4182-T always produced a 2:1 (SDR:FDR) ratio, however, H175,
had a 1:1 (SDR:FDR) ratio. These observed ratios may be
important if one mode of 2n gamete formation from the
dihaploid is desired over another. 4182-T produces
approximately one-third FDR-derived progeny out of the total

compared to one-half with H175 (Table 1-15).

45

Table 1-15. Percentage of FDR- and SDR-derived
progeny in 2x-4x crosses.

 

 

 

 

Parents Percent‘
Haploid Tetraploid FDR SDR
4182-T N00277-2 34 66
4182-T ND860-2 40 60
4182-T Lemhi Russet 32 68
4182-T Ranger Russet 31 69
4182-T Yukon Gold 32 68
H175 NDDZ77-2 55 45
H175 Yukon Gold 47 53

 

‘ Based on Pym-2 isozyme separation.

46
The variability of ratios between genotypes suggests that
the formation of FDR and SDR gametes ratios are dependent on
genotype; however, more crosses are needed to support this
observation.

In our study we saw that the yields of both FDR-derived
and SDR-derived progenies approached the midparent value for
both papulations. Yield reductions were seen each year but
the 2x-4x progenies tended toward the midparent values.
There were no trends in the distribution of values for total
yield and specific gravity for either population. A tighter
distribution might be expected with a higher percentage of
parental genome transferred intact resulting in a more
uniform gamete production in the FDR-derived tetraploid
population. The lack of consistent difference in
distribution between FDR- and SDR-derived progeny also
supports the idea of inbreeding in the dihaploid.

Performance of the 2x-4x progeny in this study support
an additive genetic model. Werner and Peloquin (1991c) and
other researchers have determined that non-additive effects
contribute to yield (Mendoza and Haynes 1974; Mendiburu and
Peloquin 1977a; Bingham et al., 1994). Therefore, our
results suggest that total yield in potato may also be due
to additive genetic effects.

The steady decline in yield over the three years for
the 4182-T x NDD277-2 population, may implicate other non-
genetic factors in the production of total tuber yield.

First of all, year to year variation in yield may be related

47
to weather conditions during the field season. During the
year with the highest yields (1991) within the 4182-T x
NDD277-2 population, total moisture was the second highest
of the three years, growing degree days accumulated were
highest and average temperature was highest of three years.
It is known that higher temperatures increase the rate of
utilization of carbohydrates produced by photosynthesis.
Consequently the amount of carbohydrates produced is
dependent on the rate of photosynthesis and length of time
that it occurs (Sieczka and Thornton 1993). Secondly, since
the experiment was conducted for three years using the
previous seasons tubers there was an opportunity for virus
accumulation. In 1992, the level of aphid infestation at
MRF was abnormally high. Large populations of aphids may
have led to increased virus vectoring. A random sample of
15 plants from each group (SDR-derived progenies, FDR-
derived progenies, 4182-T and NDD277-2) showed high virus
titers (data not shown) at harvest in 1993 according to DAS-
ELISA (Agdia, Inc., Elkhart, Indiana) results. Since it is
known that virus will lower yield, the virus titer buildup
in these populations may have reduced overall productivity.
With the virus problem widespread, we can assume that it
affected the total field experiment randomly. Despite virus
infection levels in the seed potatoes, there was never any
significant difference between SDR- and FDR-derived
progenies.

Fruit set in the 2x-4x crosses was limited (data not

48

shown) to the first half of April during the crossing
season. It has been observed in some studies that the
production of 2n eggs is dependent on temperature and
photoperiod (Hermsen 1984; Mooney and Peloquin 1992; E.
Jongedijk personal communication). Although the greenhouse
temperature was maintained at a maximum of 25°C during the
winter months, the daily maximum temperature climbed higher
as summer approached in spite of greenhouse shading. These
temperature spikes may have been a factor in fruit abortion
in the majority of crosses.

Through the use of dihaploids producing both FDR and
SDR 2n eggs we provided some insights into the question of
heterosis and the theory of maximum heterozygosity in

autopolyploids.

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Busbice, T. and C. Wilsie. 1966. Inbreeding depression and
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Medicago sativa L. Euphytica. 15:52-67.

Chujoy, J. and S. Peloquin. 1986. Tuber yields of 2x and 4x
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Concillo, L and S.J. Peloquin. 1991. Evaluation of the 4x-2x
breeding scheme in a potato breeding program adapted to
local conditions. Journal of Genetics and Breeding.
45:13-18.

Darmo, E. and S.J. Peloquin. 1990. Performance and stability

of nine 4x clones from 4x-2x crosses and four
commercial cultivars. Potato Research. 33:357-365.

49

50

Darmo, E. and S.J. Peloquin. 1991. Use of 2x TUberosum
haploid-wild species hybrids to improve yield and
quality in 4x cultivated potato. Euphytica. 53:1-9.

De Jong, H. and P. Rowe. 1971. Inbreeding in cultivated
diploid potatoes. Potato Research. 14:74-83.

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Dewey, D. 1966. Inbreeding depression in diploid,
tetraploid, and hexaploid crested wheat grass. Crop
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Douches, D. and C. Quiros. 1987. Use of 4x-2x crosses to
determine gene-centromere map distances of isozyme Loci
in Solanum species. Genome. 29:519-527.

Douches, D. and C. Quiros. 1988a. Genetic recombination in a
diploid synaptic mutant and a Solanum TUberosum x S.
Chacoense diploid hybrid. Euphytica. 38:247-260.

Douches, D.S. and C.F. Quiros. 1988b. Genetic strategies to
determine the mode of Zn egg formation in diploid
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Douches, D.S., B. Schroeter, K. Ludlam and K. Hicks. 1989.
Allelic diversity among the Solanum species, sect.
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Douches, D. and K. Ludlam. 1991. Electrophoretic
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Douches, D., D. Maas, K. Jastrebski and R. Chase. 1996.
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Dunbier, M and E. Bingham. 1975. Maximum heterozygosity in
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Grun, P, C. Ochoa and D. Capage. 1977. Evolution of
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Hawkes, J. 1990. The Potato: Evolution, biodiversity 8
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Hermundstad, S.A. and S.J. Peloquin. 1985. Germplasm
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Hokanson, K. 1995. The consequences of polyploidy on
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Hougas, R. and S. Peloquin. 1958. The potential of potato
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Howard, H. 1970. Genetics of the potato Solanum tuberosum L.
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Iwanaga, M. 1984. Discovery of a synaptic mutant in potato
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Iwanaga, M. and S. Peloquin. 1979. Synaptic mutant
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Kotch, G., R. Ortiz and S. Peloquin. 1992. Genetic Analysis
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Mendiburu, A., S. Peloquin and D.W.S. Mok. 1974. Potato
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Mendiburu, A. and S. Peloquin. 1977a. Bilateral sexual
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Mendiburu, A. and S. Peloquin. 1977b. The significance of Zn
gametes in potato breeding. Theoretical and Applied
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Mendiburu, A. and S. Peloquin. 1979. Gene-centromere mapping
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52

Mendoza, H.A. and F.L. Haynes, 1973. Some aspects of
breeding and inbreeding in potatoes. American Potato
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Mendoza, H.A. and F.L. Haynes, 1974a. Genetic basis of
heterosis for yield in the autotetraploid potato.
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Mendoza, H. A. and F. L. Haynes. 1974b. Genetic relationship
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HortScience. 9(4):328-330.

Mok, D. and S. Peloquin. 1975a. Breeding value of 2n pollen
(diplandroids) in tetraploid x diploid crosses in
potatoes. Theoretical and Applied Genetics. 46:307-314.

Mok, D and S. Peloquin. 1975b. Three Mechanisms of 2n Pollen
Formation in Diploid Potatoes. Canadian Journal of
Genetics and Cytology. 17:217-225.

Mooney, J. and S. Peloquin. 1992. 2n pollen production in 2x
clones grown in greenhouse and field locations and seed
set in 4x x 2x crosses. Report to the NCR-84 Potato
Genetics Technical Committee. Chicago, Il. 12p.

Ortiz, R. and S. Peloquin. 1991. Breeding for 2n egg
production in haploid x species 2x potato hybrids.
American Potato Journal. 68:691-703.

Ortiz, R.,S. Peloquin, R. Freyre and M. Iwanaga. 1991.
Efficiency of potato breeding using FDR 2n gametes for
multitrait selection and progeny testing. Theoretical
and Applied Genetics. 82:602-608.

Peloquin, S. 1983. Genetic engineering with meiotic mutants.
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Peloquin, S., G. Yerk, J. Werner and E. Darmo. 1989. Potato
breeding with haploids and 2n gametes. Genome 31:1000-
1004.

Ramanna, M. 1979. A re-examination of the mechanisms of 2n
gamete formation in potato and its implications for
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Rice, J. and J. Dudley. 1974. Gene Effects Responsible for
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53

Rhodes, M. and E. Dempsey 1966. Induction of chromosome
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Rowe, P. R. 1967a. Performance of diploid and vegetativley
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Rowe, P. R. 1967b. Performance and Variability of Diploid
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Stelly, D.and S. Peloquin. 1986. Formation of Zn
megagametophytes in diploid tuber-bearing Solanums.
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Tai, G. 1987. The genetic consequences of Zn gametes in
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Uijtewaal, 8., E. Jacobsen and J. Hermsen. 1987. Morphology
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753.

Watanabe, M., S. Peloquin and M. Endo. 1991. Genetic
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Werner, J. and S. Peloquin. 1987. Frequency and mechanisms
of Zn egg formation in haploid tuberosum-wild species
F1 hybrids. American Potato Journal. 64:641-654.

Werner, J. 1989. 2n eggs in diploid potatoes: occurrence,
cytology, genetics and breeding value. PhD. Thesis.
University of Wisconsin-Madison. pp. 1-100.

Werner, J.E. and S.J. Peloquin. 1990. Inheritance and two
mechanisms of Zn egg formation in 2x potatoes. Journal
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Werner, J. and S. Peloquin. 1991a. Potato haploid
performance in 2x-4x crosses. American Potato Journal.
68:801-811.

Werner, J.E. and S.J. Peloquin. 1991b. Occurrence and
mechanisms of 2n egg formation in 2x potato. Genome.
34:975-982.

54

Werner, J. and S. Peloquin. 1991c. Significance of allelic
diversity and Zn gametes for approaching maximum
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Werner, J.E., D.S. Douches and R. Freyre. 1991. Use of half-
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hybrids. Canadian Journal of Genetics and Cytolology.
6:393-402.

CHAPTER II

USE OF THE LASER SCANNING CONFOCAL
MICROSCOPE TO EXAMINE
MEGASPOROGENESIS IN POTATO

INTRODUCTION

Cytological analyses have been valuable in potato (S.
tuberosum) and related diploid species for studying the
mechanisms of 2n gamete formation in both microsporogensis
and megasporogenesis. Initial efforts focused on
microsporogensis because of the ease of examination.
Megasporocyte meiosis in synaptic mutants of Solanum sp.
were largely unstudied, with conformation of the existence
of mutants relying the upon success of pollinations (Stelly
et al., 1984). The lack of early cytological information is
largely due to laborious preparation procedures involved
with viewing ovules. Early studies of Solanum sp. ovule and
megagametophyte development required fixing of the ovule
followed by imbedding in paraffin (Herr 1971).

The embedding-sectioning technique has several
limitations. First, the process of producing slides is
tedious and time consuming for large studies. Second,
because of physical sectioning, 3-D structures are
distributed over several slides. Final interpretation of
the object requires meticulous reconstruction of multiple
sections. Stelly et al., (1984) developed a high contrast
stain-clearing technique for Solanum sp. which negated the

need for embedding.

56

57

Recent advances in technology have provided researchers
in the field of microscopy with some very powerful resources
(Czymmek et al., 1994). Confocal microscopy is not a new
discovery, but combined with advanced laser technology,
laser scanning confocal microscopy (LSCM) becomes a valuable
tool for examination of female gametophytes (Frederikson
1990, 1991, 1992). The confocal ability of the LSCM allows
extremely detailed images to be gathered from within a cell
without physical sectioning. Confocal imaging provides non-
destructive optical sections similar to that of physical
sections, but without destroying the object. Since there is
no damage to the tissue, images obtained from the laser
scanning confocal microscope are not altered by physical
sectioning and will retain true-to-life aspects. Research
in the field of developmental biology has expanded due to
the availability of the LSCM (Paddock 1994).

The objective of this research project is to examine
megasporogenesis in potato (Solanum sp.) through the use of
LSCM. Laser scanning confocal microscopy provides an
excellent opportunity to study megasporocyte meiosis with-

out traditionally tedious techniques.

58

LITERATURE REVIEW

CLEARING TECHNIQUES

In attempts to avoid imbedding and sectioning
processes, many ovule clearing techniques were developed. A
10% sucrose solution was used by Poddubnaya-Arnoldi (1960)
to clear ovules in several species of orchid. An ovule
clearing technique developed by Herr (1971) included lactic
acid, chloral hydrate, phenol, clove oil and xylene. Ovules
were placed in the 2:2:2:2:1 clearing solution for 24 hours,
then placed between a coverslip and slide with pressure
applied gradually. Herr's technique allows the ovule to
break apart without disrupting the integrity of the
individual cells. Herr's clearing method usually required 4
days to perform from fresh flower to prepared ovary tissue.
Stelly (1984) followed with a method that utilized Mayer's-
hemalum followed by clearing. Mayer’s-hemalum is a positive
stain specific for chromatin and nucleoli. Stelly's
protocol was a modification from Herr's in that the complex
solution was replaced by methyl salicylate (oil of
wintergreen) for clearing. A new step in Stelly's procedure
was to utilize whole ovules. The use of whole ovules
reduces the number of lost and damaged ovules. Stelly's

process also requires five days to fix and clear ovule

59
tissue for use. Jongedijk (1987a) improved on the clearing
method by inserting ovules into pure methyl salicylate for
30 minutes after 24 hours in a fixing solution. Jongedijk's
protocol reduced the time required to approximately 1 1/2
days. Observations were performed on non-squashed whole
ovules. Jongedijk (1987b) further modified the process by
using pectinase to degrade cell walls to separate the cells,
followed by acetocarmine stain. Jongedijk’s pectinase
protocol was shown to be very useful in viewing species with

small megasporocytes and small chromosomes such as potato.

CONFOCAL PRINCIPLES AND LSCM

There are many methods of viewing objects through a
light microscope. One of these is through the use of a
confocal microscope. Confocal microscopy images the same
plane as the illuminated plane. Confocal is defined as
having the same foci, meaning all out of focus light from
above and below the focal plane is excluded from the final
image. Advancement of laser technologies has allowed for
the refinement of stage-scanning confocal microscopes
(Minsky 1988). A laser creates a uniform, monochromatic,
coherent beam of light. The attributes of laser light
combined with the confocal ability of microscopes gives the
laser scanning confocal microscope (LSCM) even more
resolution. The LSCM can produce several types of images:
laser transmitted, reflected and fluorescent images. An

example of the light path in a LSCM is shown in Figure 2-1.

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61
The laser is focused in a plane within the sample by the
objective lens. Fluorescent light is emitted from the
illuminated spot, some of which will return through the
objective. As the light is returned through the objective
lens the dichroic mirror redirects it to a collecting lens.
Returning light is focused by the collecting lens toward a
pinhole in a metal plate. In-focus light from the image
that passes through the pinhole is allowed to expand and is
then detected by a photomultiplier tube. All of the in-
focus light from the image is passed through the pinhole,
whereas most of the out-of-focus light is excluded. Out-
of-focus light has it focal point either in front or behind
the pinhole, only in-focus light can pass through. A
confocal pinhole can remove up to 95% of the out-of-focus
light. As the laser scans across the image, light from a
number of areas is gathered and processed into an image.
The image is usually 512 x 512 or 1024 x 1024 pixels in size
(Czymmek et al., 1994).

The LSCM has the ability to produce optical sections
with enhanced resolution. Optical sections are formed from
light coming from the focal plane and are similar to
mechanical sections. Along with the ability to do
horizontal sectioning, the LSCM can collect images on the z
axis. For example, an object can be partitioned into a
series of optical sections at preset intervals and

thicknesses.

62
The resolution of the microscope is dependent on
several factors which include, but are not limited to, the
objective’s numerical objective (NA), length of light path,
the emission wavelength of the selected laser, the laser
spot size and sensitivity of the detector. The spot size
for a 488-nm laser with a high NA (1.4) is about 250 nm in
diameter (Bertero 1990; Czymmek et al., 1994). The video
monitor may also play a role in the resolution of the final
image. The number of pixels available on the video monitor

for viewing may be the limiting factor in resolution.

APPLICATION OF THE LSCM

The flexibility of the LSCM in different research areas
has been demonstrated (Carlsson 1990; Boyde 1994; Czymmek et
al., 1994; Paddock 1994). The optical sectioning capability
combined with the z-series ability is the basis of more
advanced capabilities found in the LSCM (Czymmek et al.,
1994). A z-series can be examined as a gallery with all of
the optical sections displayed side by side. The LSCM has
the ability to recombine the z-series into stereo pairs.
Multiple z-series can also be transferred to a graphics
workstation for 3-D reconstruction and production of
rotational models.

In 1990, Frederickson described the use of LSCM to
observe whole orchid ovules. Ovules were fixed in FPAm
(formalin:propionic acid: 50% ethanol, 5:5:90) for 24 hours.

To make the ovules transparent a few drops of Herr's

63

clearing fluid was applied (Herr, 1971). Prepared ovules
were viewed under a 488 nm laser which caused the ovules to
autofluoresce. Fredrickson (1990, 1991, 1992) found that
optical sections produced by the LSCM are thin and without
artifacts caused by embedding, cutting or staining.

Three-dimensional reconstructions have been performed
using LSCM optical sections (Sheppard et al., 1990; Bertero
et al., 1990). van Spronsen et al., (1989) utilized the
LSCM to make optical sections of chloroplast and then made
3-D reconstructions to explore the structure. Easily
assembled three-dimensional rotational models allow for the
exploration of the chloroplast and other structures with

little effort.

64

MATERIALS AND METHODS

1. PLANT MATERIAL

Four diploid potato clones were used for the
cytological analysis. H175 and 4182-T are dihaploids of S.
tuberosum while 84810 is a selection from S. phureja.
W5295.7 is a hybrid between S. tuberosum and S. phureja.
H175, 4182-T and W5295.7 were supplied by Drs. 8. Peloquin
and J. Werner, University of Wisconsin, Madison. H175 and
4182-T were previously identified as producers of 2n eggs
(Werner and Peloquin 1991c) whereas 84810 produced 2n pollen
by the parallel spindle mechanism. W5295.7 produces both 2n
eggs and Zn pollen. Sprouting tubers were placed on petri
dishes in three gallon plastic pots with Baccto mix in the
greenhouse in January, using high pressure sodium lights (16
hr daylength) as supplemental lighting. To enhance the
flowering period stolons were removed using a modification
of the "brick technique". Plants were thinned to 1-2 stems,
trimmed weekly and allowed to flower. The nighttime

greenhouse temperature was maintained at 25%L

2. BUD PREPARATION
Unopened flower buds of various maturity stages were

collected from the greenhouse during the months of February

65
through April. The size range of the flower buds was 5 mm to
1 mm. The flower buds were fixed for 24 hours in Farmers

solution (3:1, 95% ethanol:glacial acetic acid).

3. STAINING PROCEDURE

Ovules were stained with ethidium bromide (EtBr) and
rinsed with distilled H20. Several different stain
concentrations (5, 1, 0.5, 0.05 mg/ml at 1, 5, 15, 45
minutes) and rinse time combinations (1, 2, 3 times the

stain period) were tried.

4. SLIDE PREPARATION

The EtBr-treated ovaries were divided in two with one
half stored in sterile deo and the remainder used for slide
study. Ovules were teased out of remaining ovary tissue and
placed into a drop of glycerol on a slide. A coverslip was

applied over the ovules with gentle pressure for viewing.

5. SLIDE VIEWING

Ovules were examined with a LSCM (Zeiss 210, Carl
Zeiss, Inc., Thornwood, NY) under differing magnifications
(5x, 10x, 40x, 40x oil and 100x oil). Ovules were viewed in
fluorescence mode using a 488 nm Argon laser in combination
with a long pass barrier filter (LP520) to remove light
below 520 nm from the image. Contrast and brightness were
adjusted until a satisfactory image was obtained.

Magnification of the scanned image from 20x to 80x was

66

through use of LSCM software. One ovule at a time was
scanned to identify stages of megasporogenesis. Ovule
samples that displayed clear images were Z-sectioned into a
40 image series. Images were printed using a video printer
(Sony, Inc., Japan), saved to disk, photographed (Matrix,
Multicolor, Orangeberg, NY) or transferred to the Silicon
Graphics Workstation (Personal Iris, Silicon Graphics, Inc.,
Mountain View, CA). The z series images were processed
using VoxelMath"‘(Vital Images, Inc., Iowa). The edited
images were then transferred to VoxelViewn‘ (Vital Images,

Inc., Iowa) to produce a 3-D rotational image.

67

RESULTS

1. STAINING AND VIEWING

Serial dilutions were performed with the EtBr and dHfi)
washes (data not shown). The best staining occurred when
the ovules were placed into 0.5 mg/ml EtBr for 15 minutes
followed by three distilled water washes of 15 min each.
The 40x oil and 100x oil objectives were found to be the
most effective to examine megasporogenesis. The 40x oil was
used for rapid scanning of the slide to identify areas
containing ovules. Once the ovules were identified the
objective was changed to 100x oil and ovules were examined

further.

2. STAGES OBSERVED

Several optical sections of whole EtBr-stained Solanum
ovules under going normal meiosis are shown in Figures 2-2
to 2-11. In Figure 2-2, an ovule from 4182-T the MMC in
interphase. The chromatin is becoming distinct around the
nucleolus. Pachynema stage of prophase shows chromosomes
more distinct and recognizable in 84810 (Figure 2-3).
Diplonema is characterized by the separation of bivalents at

points along their length (Figure 2-4). During diakinesis

68

Figure 2-2. Ovule from 4182-T in
interphase. Nucleolus is visible and
the chromatin is becoming distinct.
Key: o=ovule, m=megaspore mother cell
and n=nucleolus. 3000x.

69

 

70

Figure 2-3. Pachynema stage of prophase
shows chromosomes more distinct and
recognizable in 84810. 9000x.

71

 

72

Figure 2-4. Diplonema is characterized
by the separation of bivalents at points
along their length. 10,000x

73

 

74
chromosomes are aligned in a circle on a plane with the
nucleolus of W5295.7 (Figure 2-5). Figure 2-6 and 2-7 are
of the same megaspore. In Figure 2-6, anaphase I,
chromosomes have begun separation to opposite poles of the
cell (W5295.7). A second optical section shows anaphase I
at a different depth in W5295.7 (Figure 2-7). A composite
made using the numerous optical sections provides a view of
telophase I stage in 4182-T (Figure 2-8). A stereo
reconstruction was made using the numerous optical sections
provides a 3-D relief view of telophase I stage in 4182-T
(Figure 2-9). Figure 2-10 shows a dyad produced from 4182-
T. One functional megaspore mother cell and three non-
functional daughter cells from 4182-T is observed (Figure 2-

11).

 

75

Figure 2-5. During diakinesis chromosomes
are aligned in a circle on a plane with
the nucleolus of W5295.7. 8000x

76

   

77

Figure 2-6. Anaphase I, chromosomes
have begun separation to opposite
poles of W5295.7. 2000x.

78

 

79

Figure 2-7. Different optical
section of anaphase I shows progression
of separating chromosomes in W5295.7.
6000x.

80

 

81

Figure 2-8. A composite

was made using the numerous optical
sections provides a view of telophase
I stage in 4182-T. 6000x.

82

 

83

Figure 2-9. A stereo reconstruction
was made using the numerous optical
sections provides a view of telophase
I stage in 4182-T. 2000x.

 

 

 

 

84

 

85

Figure 2-10. Dyad produced from 4182-T.
8000x.

 

 

 

 

86

 

87

Figure 2-11. Functional megaspore
and three non-functional daughter
cells from 4182-T is observed.
8000x.

 

88

 

89

DISCUSSION

The ease by which Solanum spp. ovules can be stained,
optically sectioned and photographed is demonstrated by our
results. A reduction in time and labor utilizing the LSCM-
Etbr method is noted over traditional physical sectioning
and squash techniques. Utilizing the LSCM-EtBr procedure,
26 hours were required for fixing, staining and final rinse.
This surpassed Stelly's protocol (1984) which required 4-5
days for fixing and viewing. Jongedijk's procedure (1987a)
required approximately the same time period for preparation
(27 hrs), but called for mounting and physical sectioning of
ovary samples. Early squash techniques impeded the
examination of small ovules by loss and destruction of
samples. Jongedijk (1987b) stated that only 40% of the
megasporocytes were usable for examination after squash
preparation techniques. The LSCM procedure compares to
Herr's clearing method (1984) in which whole ovules were
processed. In the LSCM-EtBr procedure no clearing technique
is applied, the confocal ability of the microscope allows
the observation of the MMC through numerous cell layers
(Figure 2-8). Optical sectioning by the LSCM does not
subject the ovule to any mechanical pressure, providing a
accurate image with excellent definition.

EtBr having an affinity for chromosomes and spindle

90
fibers provided an ideal stain for this technique.
Preliminary surveys (data not shown) of fixed buds showed a
faint autofluorescence similar to that noted by Fredrikson
(1992), but not sufficient to view chromosomes clearly.
Staining was accomplished with a single 0.5 mg/ml EtBr stain
and triple rinse in sterile ddeo. Using higher
concentrations stained the outer layers heavily, but inner
layers had absorbed little EtBr. Stronger concentrations
needed less time to stain, but required longer rinse times
to reduce background fluorescence and bleeding of EtBr into
the glycerol. Individual ovules physically separated from
the ovary could be stained using a lower concentration of
EtBr for a shorter time with minimal background
fluorescence. Since the EtBr-stained cellular material
fluoresces brightly on a dark background, the images from
the LSCM are in reverse of conventional images. Whereas
Fredrikson (1992) reversed the LSCM images to make them more
comparable to microscope images, ours are not. It may be
possible to utilize photophores that would stain selectively
for different parts of the cell (Entwistle and Noble 1992),
and thus acquire more information on the activities of the
cellular components during meiosis.

Optical images obtained by the LSCM can be manipulated
in many ways before saving to provide the best definition.
The contrast and brightness of the image can be adjusted as
well as enhanced using numerous digital filters. To obtain

desired levels of contrast for Solanum ovules Stelly et al.

91
(1984) was required to use various film development regimes.
To obtain similar results with the LSCM, contrast
adjustments are made via a knob, providing an instant
change, as compared to a tedious film development schedule.
Since microscope images are stored digitally on magnetic
media they can be recalled at any time for manipulation or
to be rephotographed. The use stored digital images is
extremely advantageous over traditional microscope
photography techniques. A photographed image is stored on a
fragile negative, if the negative is scratched the image
quality is severely reduced. Subsequent negatives can be
reproduced from existing pictures, but they will be lacking
the quality of the original. An image saved digitally can
be recalled and reproduced with the original clarity.
Several desktop imaging programs can be used to adjust the
optical images away from the microscope. Digital
microscope images can be imported into slide preparation
programs to construct presentations. These images can be
distributed as tools for classroom instruction to anyone
with a desktop computer for viewing.

The LSCM has the ability to create stereo pairs from a
z-series of optical sections from a single object.
Stereoscopic images viewed with a stereoscope produce a 3-D
effect which allow for the viewing of objects in relief.
Subsequent transfer of a z-series to the graphics
workstation can be completed quickly. Subsequent reassembly

of unaltered optical sections is done using VoxelView“fl

 

92
VoxelViewm can quickly produce a 3-D rotational model. If
changes were made to the images such as trimming, altering
the opacity of an object, adding color or masking; the final
product may require several additional hours of work.
Reconstructions of rotational 3-D images from ovule optical
sections present yet another method to examine
megasporogenesis. The method of reconstruction used with
the potato ovule (data not shown) was similar to that of van
Spronsen (1989). van Spronsen used 16 optical sections in
the reconstruction, whereas the potato ovules required at
least 40 sections. The reassembled 3-D image can be
adjusted to emphasize certain areas of the ovule that may
not have been apparent with 2-D studies.

Meiotic cells were easily to distinguish from the
surrounding somatic cells by their large size and
distinctive appearance (Figures 2-2, 2-6). All stages of
Meiosis I were readily identified in ovary samples. LSCM
images of meiosis were similar in structures to that of
Jongedijk’s (1987a). Jongedijk (1987a) noted that within
plant species such as potato, female meiosis is highly
synchronized, entailing a search of numerous ovaries to
identify all stages. In this study, meiosis II stages were
not identified and may be more transient and difficult to
detect. The appearance of Zn eggs has not been confirmed,
other than the possible production of a dyad (Figure 2-10).
Due to the stage of the megasporocyte it was not possible to

determine whether the megaspore was FDR- or SDR- derived.

93
Production of 2n eggs in Solanum is under genetic control,
however the environment can influence frequency. It was
noted that production of 2n eggs might be dependent on
temperature and photoperiod conditions of the source plant
(Mooney and Peloquin 1992; E. Jongedijk personal
communication).

The use of the LSCM-Etbr technique provide an excellent
tool to expand exploration of other species with small
chromosomes like potato. An immense savings in preparation
time allows for the quick examination of large numbers of
ovules. The resulting images are very clear and suitable
for many uses. The LSCM advances our ability to examine
megasporogenesis in potato efficiently and effectively and

justifies further experimentation.

 

LITERATURE CITED

Bertero, M., P. Boccacci, G. Brakenhoff, F. Malfanti and H.
van der Voort. 1989. Three-dimensional image
restoration and super resolution in fluorescence

confocal microscopy. Journal of Microscopy. 157:pt 1:3-
20.

Boyde, A. 1994. Bibliography on confocal microscopy and its
applications. Scanning. 16:33-56.

Brakenhoff, G. 1979. Imaging modes in confocal scanning
light microscopy (CSLM). Journal of Microscopy. 117 pt
2:233-242.

Carlsson, K. 1990. Scanning and detection techniques used in
a confocal scanning laser microscope. Journal of
Microscopy. 21-27.

Czymmek, K., J. Whallon and K. Klomparens. 1994. Review:
Confocal microscopy in mycological research.
Experimental Mycology. 18:275-293.

Entwistle, A. and M. Noble. 1992. The use of lucifer yellow,
FITC, TRITC, RITC and texas red for dual
immunofluorescence visualized with a confocal scanning
laser scanning microscope. Journal of Microscopy.
168(3):219-238.

Fredrikson, M. 1992. The development of the female
gametophyte of Epipactis (Orchidaceae) and its
inference for reproductive ecology. American Journal of
Botany. 79(1):63-68.

Henstra, S.,L. Bremer and A. Boekestein. 1989. Applications
of the confocal scanning laser microscope in
agricultural research. Ultramicroscopy. 31:467-468.

Herr, J. 1971. A new clearing-squash technique for the study
of ovule development in angiosperms. American Journal
of Botany. 58(8):?85-790.

Inoue, 8. 1989. Foundations of confocal scanned imaging in
light microscopy. In Handbook of biological confocal
microscopy. J. Pawlye, Ed. pp 1-13. Plenum, New York.

Jongedijk, E. 1987a. A rapid methyl salicylate clearing
technique for routine phase-contrast observations on
female meiosis in Solanum. Stain Technology 146(2):157-
162.

94

 

95

Jongedijk, E. 1987b. A quick enzyme squash technique for
detailed studies on female meiosis in Solanum. Stain
Technology. 62(3):135-141.

Maheshwari, P. 1950. An introduction to the embryology of
angiosperms. McGraw-Hill. New York.

Minskey, M. 1988. Memoir on inventing the confocal scanning
microscope. Scanning. 10:128-138.

Mooney, J. and 8. Peloquin. 1992. 2n pollen production in 2x
clones grown in greenhouse and field locations and seed
set in 4x x 2x crosses. Report to the NCR-84 Potato
Genetics Technical Committee. Chicago, Il. 12p.

Paddock, 8. W., 1994. To boldly glow.....Applications of
laser scanning confocal microscopy in developmental
biology. BioEssays. 16:357-365.

Poddubnaya-Arnoldi, V. 1963. The culture of some orchid
ovules on an artificial nutritive medium. Journal of
Indian Botany. 42:180-184.

Sheppard, C. and C. Cogswell. 1990. Three-dimensional image
formation in confocal microscopy. Journal of
Microscopy. 179-194.

Stelly, M., S. Peloquin, R. Palmer and C. Crane. 1984.
Mayer’s hemalum-methyl: a stain clearing technique for
observations within whole ovules. Stain Technology.
59(3):155-l61.

van Spronsen, E., V. Sarafis, G. Brakenhoff, H. van der Vort
and N. Nanninga. 1989. Three-dimensional structure of
living chloroplast as visualized by confocal scanning
laser microscopy. Protoplasma. 148:8-14.

APPENDIX A

 

 

97

 

 

 

 

 

 

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98

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99

Table A-3. Summary of soil test for the
general plot area for the growing season at Montcalm
Research Farm, Montcalm, MI.

        
   
   

 

 

 

 

 

 

 

 

 

====-==
pH ago, 1:20 c. s . c
6.1 449 219 800 152 5.3 me/100 -
5.6 383 152 610 124 5.8 me/100 -
6.2 395 177 762 137 2.7 me/lOO -
“2.7 me/100 g”

 

 

Year

100

The four year summary of growing
degree days (base 10 C‘) during the season at
Montcalm Research Farm, Montcalm, MI.

June

July

August

September

 

1991
1992
1993
1994

 

1014

 

1632

 

 

2185

 

2491
1956
2153
2148

 

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